As a result of the recent studies, it has been clarified that hepatitis C virus is classified into a large number of types, depending on genotype or serotype. In accordance with the phyloanalysis method of Simmonds et al. using the nucleotide sequences of HCV strains, which is presently being used as a mainline HCV genotype classification method, HCV is classified into the following 6 types: genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3a, and genotype 3b (Non-Patent Document 1). These types are further classified into several subtypes. The nucleotide sequences of the full-length genomes of a plurality of genotypes of HCV have also been determined (Patent Document 1 and Non-Patent Documents 2 to 4).
HCV causes chronic hepatitis as a result of persistent infection. A main cause of chronic hepatitis, which is recognized on a global scale, is persistent HCV infection. As a matter of fact, approximately 50% of persistently infected patients develop chronic hepatitis, and approximately 20% of the patients shift to hepatocirrhosis over 10 to 20 years. Moreover, some patients thereof develop fatal pathologic conditions such as liver cancer.
At present, the main treatments for hepatitis C include the use of interferon-α or interferon-β, and the combined use of interferon-α with ribavirin, which is a purine-nucleoside derivative. However, although these treatments are performed on patients, the therapeutic effects thereof are observed only in approximately 60% of such patients. If the treatments are terminated after such therapeutic effects have been obtained, more than half of the patients develop recurrent disease. It has been known that the therapeutic effects of interferon depend on the genotype of HCV. That is, it is said that the effects of interferon are low on genotype 1b and that the effects thereof are high on genotype 2a (Non-Patent Document 5). Moreover, the substrate specificity of protease of HCV is different depending on genotype. The inhibitory activity of an inhibitor developed using NS3 protease of genotype 1b is 50 times or more inferior to those developed using NS3 proteases of other genotypes (Non-Patent Document 6). Accordingly, in order to develop an HCV therapeutic agent with efficiency, it is required to develop the agent, while confirming the reactivity of each of the genotypes of HCV.
Recently, an HCV subgenomic RNA replicon has been produced as RNA derived from HCV which can be autonomously replicated (Patent Documents 2 and 3 and Non-Patent Documents 7 to 9). Thereby, it became possible to analyze HCV replication mechanisms, using cultured cells. Such an HCV subgenomic RNA replicon is produced by substituting a structural protein existing downstream of HCV IRES, in the 5′ untranslated region of HCV genomic RNA, with a neomycin resistance gene and EMCV-IRES that is ligated downstream thereof. This RNA replicon was introduced into human liver cancer cells Huh7, and the cells were then cultured in the presence of neomycin. As a result, it was demonstrated that the RNA replicon autonomously replicates in Huh7 cells. Moreover, it was also demonstrated that several HCV subgenomic RNA replicons autonomously replicate in cells other than Huh7, such as human cervical cancer cells HeLa, or human liver cancer cells HepG2 (Patent Document 3).
However, such HCV intracellular RNA replication systems have been produced for limited genotypes, or rather, such systems have been produced only using genomic RNAs of a limited number of HCV strains. Thus, with regard to HCV having a large number of genotypes, it is extremely difficult to analyze differences in therapeutic effects of the developed HCV therapeutic agents that are caused by differences in the genotypes of the above agents. Such an RNA replicon is an experimental system, which is only useful for evaluating the replication of virus RNA during the growth and replication process of an HCV virus. Hence, it is impossible for such an RNA replicon to evaluate processes, such as formation of HCV virus particles in an infected cell, the release thereof out of the cell, or infection of a new cell.
Currently, application of a method for evaluating such processes as formation of HCV virus particles, the release thereof out of the cell, and infection of a new cell is limited to an experimental system using animals such as chimpanzees (Non-Patent Document 10). However, such an experimental system, in which living animal bodies are directly used, involves complicated operations, and thus it is extremely difficult to conduct analyses with such an experimental system. Accordingly, in order to analyze such processes as formation of HCV virus particles, the release thereof out of the cell, and infection of a new cell, or in order to develop an anti-HCV agent using inhibition of such processes as an action mechanism, it is necessary to construct an extremely simplified experimental system capable of replicating such processes; namely, an HCV virus particle replication system using a cultured cell system.
If it became possible to stably supply HCV virus particles from such a cultured cell system, a virus could be attenuated, or a noninfectious HCV virus could be produced by means based on molecular biology, thereby using such viruses as vaccines. However, since HCV protein sequences differ depending on genotype, the antigenicity of HCV also differs depending on genotype. In fact, the presence of various genotypes constitutes a significant impediment to the production of HCV vaccines (Non-Patent Document 11). Accordingly, in order to efficiently produce HCV vaccines as well, it has been desired that HCV virus particles with various genotypes be stably produced in a cultured cell system.
It has been known that HCV is a spherical particle with a size between 55 and 65 nm, which exists in the blood of a patient infected with HCV. As a method for purifying HCV existing in human serum, affinity chromatography using lectin (Non-Patent Document 12) and chromatography using heparin (Non-Patent Document 13) have been known. However, by these methods, only less than 1 ml of virus can be purified at a concentration of approximately 1 M copies/ml. Thus, these methods are not industrially applicable.
Several methods for purifying virus particles other than HCV have been created to date (Patent Documents 4, 5, and 6, for example). However, as is clear from these publications, virus particles have various properties, and thus the particles give no useful information regarding an optimal method for purifying human hepatitis C virus. Patent Document 7 discloses that human hepatitis A virus, which is also a hepatitis virus, can be purified by eliminating DNA according to anion exchange chromatography. However, although hepatitis A virus is also a hepatitis virus, it is a virus having DNA as a gene. As is clear from the fact that hepatitis C virus has RNA as a gene, there are no relevant similarities between hepatitis A virus and hepatitis C virus, and thus no information is given regarding relevant purification methods. In order to use human hepatitis C virus particles as vaccines or the like in the industrial field in the future, it is required to highly purify such particles in high volume. Under such circumstances, the development of a purification method is anticipated.
[Patent Document 1]    JP Patent Publication (Kokai) No. 2002-171978 A
[Patent Document 2]    JP Patent Publication (Kokai) No. 2001-17187 A
[Patent Document 3]    WO2004/104198A1
[Patent Document 4]    Japanese Patent No. 3313117
[Patent Document 5]    JP Patent Publication (Kohyo) No. 2002-503484 A
[Patent Document 6]    JP Patent Publication (Kohyo) No. 2000-510682 A
[Patent Document 7]    JP Patent Publication (Kokoku) No. 6-48980 B (1994)
[Non-Patent Document 1]    Simmonds P. et al., Hepatology, 10 (1994) pp. 1321-1324
[Non-Patent Document 2]    Choo Q. L. et al., Science, 244 (1989) pp. 359-362
[Non-Patent Document 3]    Okamoto H. et al., J. Gen. Virol., 73 (1992) pp. 673-679
[Non-Patent Document 4]    Mori S. et al., Biochem. Biophis. Res. Commun. 183 (1992) pp. 334-342
[Non-Patent Document 5]    Yoshioka K. et al., Hepatology, 16 (1992) pp. 293-299
[Non-Patent Document 6]    Thibeault D. et al., J. Virol., 78 (2004) pp. 7352-7359
[Non-Patent Document 7]    Blight et al., Science, 290 (2000) pp. 1972-1974
[Non-Patent Document 8]    Friebe et al., J. Virol., 75 (2001) pp. 12047-12057
[Non-Patent Document 9]    Kato T. et al., Gastroenterology, 125 (2003) pp. 1808-1817
[Non-Patent Document 10]    Kolykhalov et al., Science, 277 (1997) pp. 570-574
[Non-Patent Document 11]    Farci P. et al., Semin Liver Dis 20 (2000) pp. 103-126
[Non-Patent Document 12]    Virology, 196 (1993) pp. 354-357
[Non-Patent Document 13]    Journal of General Virology 86 (2005) pp. 677-685